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Abstract:

Bispecific antisense oligonucleotides which consist essentially of a
sequence of bases that is complementary to portions of both the gene
encoding human IGFBP-2 and the gene encoding human IGFBP-5 are useful in
as antisense therapeutics in the treatment of endocrine-regulated
cancers.

20. The method of claim 19 wherein in the exposing step, the cells are
exposed to a pharmaceutical composition made by combining the bispecific
antisense oligonucleotide with a pharmaceutically acceptable carrier for
intravenous, intraperitoneal, subcutaneous or oral administration.

21. The method of claim 20, wherein the oligonucleotide is modified to
increase the stability of the antisense in vivo.

22. The method of claim 21, wherein the oligonucleotide is a
phosphorothioate derivative.

23. The method of claim 21, wherein the oligonucleotide has
2'-O-methoxyethyl substitutions on the backbone.

24. The method of claim 19, wherein the oligonucleotide is modified to
increase the stability of the antisense in vivo.

25. The method of claim 24, wherein the oligonucleotide is a
phosphorothioate derivative.

26. The method of claim 24, wherein the oligonucleotide has
2'-O-methoxyethyl substitutions on the backbone.

Description:

[0001] This application claims the benefit of U.S. Provisional Application
No. 60/350,046, filed Jan. 17, 2002, which is incorporated herein by
reference.

BACKGROUND OF THE INVENTION

[0002] This present application relates to bispecific antisense
olignucleotides that inhibit IGFBP-2 and IGFBP-5 and methods of using
same in the treatment of endocrine-regulated tumors (for example, breast,
prostate, ovarian and colon cancers).

[0003] Prostate cancer is the most common cancer that affects men, and the
second leading cause of cancer deaths in men in the Western world.
Because prostate cancer is an androgen-sensitive tumor, androgen
withdrawal, for example via castration, is utilized in some therapeutic
regimens for patients with advanced prostate cancer. Androgen withdrawal
leads to extensive apoptosis in the prostate tumor, and hence to a
regression of the disease. However, castration-induced apoptosis is not
complete, and a progression of surviving tumor cells to
androgen-independence ultimately occurs. This progression is the main
obstacle to improving survival and quality of life, and efforts have
therefore been made to target androgen-independent cells. These efforts
have focused on non-hormonal therapies targeted against
androgen-independent tumor cells, however as of a 1998 report, no
non-hormonal agent had improved survival. Oh et al., J. Urol 160:
1220-1229 (1998) Alternative approaches are therefore indicated.

[0005] The biological response to IGF's is regulated by various factors,
including IGFBPs. To date, six IGFBPs have been identified whose function
is believed to involve modulation of the biological actions of the IGFs
through high affinity interactions. Rajaram et al., Endocrin. Rev. 18:
801-813 (1997). However, some evidence suggests biological activity for
IGFBPs that are independent of IGFs, Id., Andress et al., J. Biol. Chem.
267: 22467-22472 (1992); Oh et al., J. Biol. Chem. 268: 14964-14971
(1993), and both stimulatory and inhibitory effects of IGFBPs on cell
proliferation have been reported under various experimental conditions.
Andress et al., supra; Elgin et al., Proc. Nat'l. Acad. Sci. (USA), 84,
3254-3258 (1987); Huynh et al., J. Biol. Chem. 271: 1016-1021 (1996);
Damon et al., Endocrinology 139: 3456-3464 (1998). Thus, the precise
function role of IGFBPs remains controversial. Because of this, while the
reported results implicate IGF in prostate and breast cancer, they do not
clearly suggest a therapeutic approach based upon this involvement.

[0006] PCT Publication WO 01/05435, which is incorporated herein by
reference, describes a method for treating hormone-regulated tumors (for
example, breast and prostatic tumors) in mammals, including humans, by
administration of an antisense oligodeoxynucleotide which is
complementary to a portion of the gene encoding IGFBP-5. PCT Publication
No. WO 02/22642, which is incorporated herein by reference, describes a
method are provided for the treatment of prostate and other endocrine
tumors in mammals, including humans, by administration of an antisense
oligodeoxynucleotide which is complementary to a portion of the gene
encoding IGFBP-2.

[0008] The present invention provides a method for treating
endocrine-regulated tumors (for example, breast, prostate, ovarian and
colon cancers) in mammals, including humans, by administration of a
bispecific antisense oligodeoxynucleotide which is complementary to
portions of both the gene encoding IGFBP-2 and the gene encoding IGFBP-5.
The administration of such an oligodeoxynucleotide was shown to reduce
levels of both IGFBP-2 and IGFBP-5 in PC3 and LNCaP cells, and the
reduction of expression of the proteins has been previously shown to
reduce proliferation of tumor cells, and also to delay the progression to
androgen independence. Thus, in accordance with the invention we provide
methods for treatment of prostate cancer in mammals, including humans,
and for delaying the progression of prostate tumors to androgen
independence comprising the step of administering to the mammal a
therapeutically effective amount of a bispecific antisense
oligodeoxynucleotide which is complementary to portions of both the
nucleic acid sequence encoding IGFBP-2 and the nucleic acid sequence
encoding IGFBP-5, and which hybridizes with such sequences to inhibit or
otherwise reduce the expression of IGFBP-2 and IGFBP-5.

[0009] The application further provides bi-specific antisense sequences
that can be used in the method of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[0010] FIGS. 1A and 1B show the amount of IGBFP-2 and IGBFP-5 expression
observed in LNCaP and PC3 cells, respectively, upon administration of the
three bispecific antisense oligonucleotide, a mismatch control (MM), or
no olignucleotide (control).

[0015]FIG. 7 shows the results of real time PCR measurements of IGFBP-2
and IGFBP-5 LNCaP/msf in mixed tumors and in intact fetal bone fragments

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides bispecific antisense
oligonucleotides which consist essentially of a sequence of bases that is
complementary to portions of both the gene encoding IGFBP-2 and the gene
encoding IGFBP-5, and that is sufficient length to act as an inhibitor of
the effective amount of IGFBP-2 and IGFBP-5 (in general at least 15
bases). As used in the specification and claims of this application, this
language means that substantially all of the antisense oligonucleotide is
complementary to a portion of each gene sequence. The invention does not,
however, exclude minor modifications in sequence, such as the addition of
one or two terminal bases, or single base substitutions which might
depart from perfect complementarity but which still function as an
inhibitor of the effective amount of IGFBP-2 and IGFBP-5.

[0017] The phrase "inhibitor of the effective amount" takes into account
the fact that antisense oligonucleotides may function by different
mechanisms. The effective amount of IGFBP-2 or IGFBP-5 is the amount that
is present in a functional state in the cell. Reduction of this amount by
administration of antisense oligonucleotides may occur through
restricting production of the IGFBP (at the transcription or translation
level) or by degrading the IGFBP at a rate faster than it is being
produced. Further, it will be appreciated that the inhibition which is
referred to is one which occurs when the IGFBP would otherwise be present
if the antisense oligonucleotide had not been administered. As discussed
below, not all cells that are potential therapeutic targets express both
IGFBP-2 and IGFBP-5.

[0018] Specific antisense oligonucleotides according to the invention
consist essentially of a series of bases as set forth in Seq. ID. No. 1
through 7. These sequences are set forth in Table 1.

[0019] The antisense oligonucleotides employed may be modified to increase
the stability of the antisense oligonucleotide in vivo. For example, the
antisense oligonucleotides may be employed as phosphorothioate
derivatives (replacement of a non-bridging phosphoryl oxygen atom with a
sulfur atom) which have increased resistance to nuclease digestion.
Increased antisense oligonucleotide stability can also be achieved using
molecules with 2-methoxyethyl (MOE) substituted backbones as described
generally in U.S. Pat. No. 6,451,991 and U.S. patent application Ser. No.
10/080,794 which are incorporated herein by reference.

[0020] The compositions of the present invention can be used for treatment
of endocrine-regulated tumors (for example, breast, prostate, ovarian and
colon cancers) in mammals, including humans, by administration of a
bispecific antisense oligonucleotide in accordance with the invention.
Administration of antisense oligonucleotides can be carried out using the
various mechanisms known in the art, including naked administration and
administration in pharmaceutically acceptable carriers. For example,
lipid carriers for antisense delivery are described in U.S. Pat. Nos.
5,855,911 and 5,417,978 which are incorporated herein by reference. In
general, the antisense is administered by intravenous, intraperitoneal,
subcutaneous or oral routes. Where the oligonucleotides are administered
in a pharmaceutically acceptable carrier, the carrier is generally free
from substances which produce toxic or other harmful reactions when
administered to humans.

[0021] The amount of antisense oligonucleotide administered is one
effective to reduce the effective amount of levels of IGFBP-2 and/or
IGFBP-5 in the endocrine-regulated tumor cell of concern. As noted above,
in the context of the present invention, applicants do not intend to be
bound by any specific mechanism by which this reduction may occur,
although it is noted that the reduction may occur as a result of reduced
expression of IGFBP-2 and -5 if the antisense molecule interferes with
translation of the mRNA, or via an RNase mediated mechanism. Furthermore,
it will be appreciated that the appropriate therapeutic amount will vary
both with the effectiveness of the specific antisense oligonucleotide
employed, and with the nature of any carrier used. The determination of
appropriate amounts for any given composition is within the skill in the
art, through standard series of tests designed to assess appropriate
therapeutic levels.

[0022] The method for treating endocrine-regulated cancer in accordance
with the invention may further include administration of chemotherapy
agents and/or additional antisense oligonucleotides directed at different
targets. For example, conventional chemotherapy agents such as taxol
(paclitaxel or docetaxel) and mitoxanthrone may be used. Similarly,
combinations of the bispecific antisense oligonucleotide of the invention
with other antisense sequences such as antisense Bcl-2 oligonucleotide,
TRPM-2 (clusterin) oligonucleotide, IGFBP-2 or IGFBP-5 oligonucleotide
may be used.

[0023] The effectiveness of the present invention is founded on solid
theoretical and experimental bases. Activation of alternative growth
factor pathways following androgen withdrawal is one mechanism mediating
androgen independent (AI) progression in advanced prostate cancer.
Insulin-like growth factor-I (IGF-I) activation is modulated by a family
of insulin-like growth factor binding proteins (IGFBPs). Although IGFBP-2
and IGFBP-5 are among the most commonly over-expressed genes in hormone
refractory prostate cancer, the functional significance of changes in
IGF-I signaling during AI progression remain poorly defined. AI
progression of prostate cancer leads to treatment resistance and
ultimately culminates in the development of bone metastases, the most
ominous sign of clinical progression in prostate cancer patients with
advanced disease. This development usually precedes death by 12-18
months. The bone environment, particularly rich in IGFBP-5, has long been
identified as an attractive "soil" for supporting prostate cancer
metastasis. The crucial IGF-1 signaling components IGFBP-2 and IGFBP-5
may not only be key to AI progression but also to the site-specific
metastasis of prostate cancer. In an attempt to address these issues, we
characterized changes in IGFBP-2 and IGFBP-5 in prostate cancer models
after androgen withdrawal and evaluated their functional significance in
AI progression using gain-of-function and loss-of-function analyses.

[0024] IGFBP-2 mRNA and protein levels increase 2-3-fold after androgen
withdrawal in LNCaP cells in vitro in LNCaP tumors during AI progression
in vivo. Increased IGFBP-2 levels after castration were also identified
using a human prostate tissue microarray of untreated and post-hormone
therapy-treated prostatectomy specimen. LNCaP cell transfectants (LNBP-2)
that stably over-expressed IGFBP-2 had a shorter cell doubling-time and a
lower rate of apoptosis in the absence of androgens. Reporter assays
demonstrated that this increased growth potential did not result from
IGFBP-2-mediated transcription of androgen-response elements. In vivo,
the LNBP-2 cell line formed significantly more tumors in castrate mice
and progressed to androgen independence more rapidly compared with a
control cell line. Antisense oligonucleotides targeting IGFBP-2 reduced
IGFBP-2 mRNA and protein expression by >70% in a dose-dependent and
sequence-specific manner. Antisense oligonucleotide-induced decreases in
IGFBP-2 reduced LNCaP cell growth rates and increased apoptosis 3-fold.
LNCaP tumor growth and serum PSA levels in mice treated with castration
plus adjuvant IGFBP-2 antisense oligonucleotide s were significantly
reduced compared to mismatch control oligonucleotides.

[0025] Similar findings were obtained with gain-of-function and
loss-of-function analyses of IGFBP-5 in IGFBP-5 expressing prostate
cancer models. The growth rates of IGFBP-5 transfected LNCaP cells were
significantly faster compared to either the parental or vector-only
transfected LNCaP cells in both the presence and absence of
dihydrotestosterone. IGFBP-5-induced increases in LNCaP cell
proliferation occurs through both IGF-I-dependent and -independent
pathways, with corresponding increases in the cyclin D1 mRNA expression
and the fraction of cells in S+G2/M phases of the cell cycle. Changes in
Akt/protein kinase B (PKB), a downstream component of
phosphatidylinositol 3'-kinase (PI3K) pathway, in the LNCaP sublines also
paralleled changes in their growth rates. Although treatment with a PI3K
inhibitor induced apoptosis in both control and IGFBP-5-overexpressing
LNCaP cells, this PI3K inhibitor-induced apoptosis was prevented by
exogenous IGF-I treatment only in IGFBP-5 transfectants, suggesting that
IGFBP-5 overexpression can potentiate the antiapoptotic effects of IGF-I.
Furthermore, tumor growth and serum PSA levels increased several fold
faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration
despite having similar tumor incidence and tumor growth rates with
controls when grown in intact mice before castration. These data suggest
that IGFBP-5 overexpression in prostate cancer cells after castration is
an adaptive cell survival mechanism that helps potentiate the
antiapoptotic and mitogenic effects of IGF-I, thereby accelerating
progression to androgen-independence through activation of the
PI3K-Akt/PKB signaling pathway. Systemic administration of IGFBP-5
antisense oligonucleotides significantly delayed time to progression to
androgen independence and inhibited growth of AI recurrent tumors.
IGFBP-5 expression in human bone was confirmed.

[0026] Inhibiting IGFBP-2 and IGFBP-5 expression using targeted antisense
technologies provides a treatment platform to delay AI progression and
bone metastasis in prostate cancer patients and patients with other
malignancies expressing these key regulators of IGF-1 signaling. By
targeting both IGFBP-2 and IGFBP-5 simultaneously with a single antisense
oligonucleotide the complications associated with use of a cocktail of
antisense oligonucleotides targeting either of these compounds separately
are avoided.

[0027] The application is further described in the following non-limiting
examples.

EXAMPLE 1

[0028] LNCaP cells were treated with treated with 500 nM concentrations of
antisense oligonucleotides of Seq. ID Nos. 1, 2 or 3 or a mismatch
control. Levels of IGFBP-2 were measured. The result are summarized in
FIG. 1A.

[0029] PC3 cells were treated with treated with 500 nM concentrations of
antisense oligonucleotides of Seq. ID Nos. 1, 2 or 3 or a mismatch
control. Levels of IGFBP-5 were measured. The result are summarized in
FIG. 1B.

EXAMPLE 2

[0030] Samples of human prostate cancer cells were sectioned and
immuno-stained to detect IGFBP-2. Tissue microarray scoring showed
expression of IGFBP-2 was essentially absent in benign, non-cancerous
tissues but increased with time to highest levels after androgen
independent was achieved. (FIG. 2)

EXAMPLE 3

[0031] Osseous metastases of human prostate cancer was evaluated in 15
specimens. Human prostate cancer expressed IGFBP-2 in very high levels,
but also expressed IGFBP-5. Human bone osteocytes expressed high levels
of IGFBP-5 and lower levels of IGFBP-2.

EXAMPLE 4

[0032] Bispecific antisense oligonucleotides were used to treat PC3, LNCaP
and bone cells at concentration of 500 nm, and the amount of inhibition
of IGFBP-2 or IGFBP-5 was measured using real time PCR. Oligonucleotides
of Seq. ID Nos. 2, 4, 5 and 7 were tested, and all were effective to
reduce the detected amount of the IGFBP measured. (FIG. 3A) Reductions of
up to 70% in IGFBP-2 levels were also observed in A549 lung cells using
500 nM of Seq. ID Nos. 4 and 5. (FIG. 3B) Seq. ID No. 4 (500 nM) was also
shown to be effective to inhibit cell growth of LNCaP cells and reduce
the cell number by more than 90%. (FIG. 3C) FIGS. 3D and E respectively
show results for inhibition of IGFBP-5 levels in PC3 cells with 500 nM of
Seq ID Nos. 1, 2, 4 5, 6 and 7; and inhibition of IGBFP-5 levels in human
fetal bone fobroblast cells with 500 nM of Seq. ID Nos. 1, 2, 4, 5, 6 and
7.

EXAMPLE 5

[0033] Real time PCR was used to measure the amount IGFBP-5 in MSF human
fetal fibroblast cells after treatment with antisense oligonucleotide and
LIPOFECTIN (4 μg/ml). Cells were plated in vitro and treated with a
four-hour pulse of 500 nM oligonucleotide followd by a 20 hour period in
normalmedium plus 5% serum. A second four-hour pulse was repeated on day
2 and the cells were counted on day 3. The most active antisense
oligonucleotides were Seq. ID Nos. 4 and 5. (FIG. 6)

EXAMPLE 6

[0034] Real time PCR was used to evaluate the amounts of IGFBP-2 and
IGFBP-5 in human bladder cancer (RT4) following treatment with varying
amounts of antisense oligonucleotides (Seq. ID Nos. 4 and 5) and 4
μg/ml LIPOFECTIN. As shown in FIGS. 4 and 5, a dose dependent response
was observed to both antisense oligonucleotides at concentrations ranging
from 50 to 500 nM.

EXAMPLE 7

[0035] Real time PCR was used to measure IGFBP-2 and IGFBP-5 in LNCaP/msf
mixed tumors and in intact fetal bone fragments. The results are shown in
FIG. 7.